Processes for the production of aminoalkyl glucosaminide phosphate and disaccharide immunoeffectors and intermediates therefor

ABSTRACT

This invention relates to processes for production of alkylamino glucosaminide phosphate compounds, and of disaccharide compounds, including various novel intermediates and intermediate processes. In one aspect, glycosyl halides are produced by reaction of an O-silyl glycoside with a dihalomethyl alkyl ether.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority of U.S. provisional application60/394,487 filed Jul. 8, 2002. Said provisional application is relatedto U.S. patent application Ser. No. 10/137,730, filed Apr. 30, 2002,which is a continuation-in-part of U.S. patent application Ser. No.10/043,486, filed Jan. 8, 2002, now U.S. Pat. No. 7,063,967, which is acontinuation-in-part of U.S. patent application Ser. No. 09/905,160,filed Jul. 12, 2001, now U.S. Pat. No. 6,764,840, which is acontinuation-in-part of U.S. patent application Ser. No. 09/439,839,filed Nov. 12, 1999, now U.S. Pat. No. 6,303,347, which is acontinuation-in-part of U.S. patent application Ser. No. 08/853,826,filed May 8, 1997, now U.S. Pat. No. 6,113,918. This application is alsorelated to U.S. patent application 09/074,720 filed May 7, 1998, nowU.S. Pat. No. 6,355,257, which is also a continuation-in-part of U.S.application Ser. No. 853,826. This application also claims priority ofU.S. provisional application 60/438,585 filed Jan. 6, 2003. All of saidpatents and applications are incorporated herein by reference, in theirtotalities.

BACKGROUND OF THE INVENTION

This invention relates to processes for the production of aminoalkylglucosaminide phosphate (AGP) and of disaccharide compounds. Suchcompounds have been found to be immunoeffectors, adjuvants for vaccinesand the like, and in addition, can possess therapeutic and/orprophylactic properties of their own. In addition, this inventionrelates to processes for the production of glycosyl halides, which canserve as intermediates in the synthesis of AGP compounds, disaccharides,and structurally related molecules.

Aminoalkyl glucosaminide phosphates are described in a number ofpatents, published patent applications, and journal articles. Suchcompounds in general have five or six acyl groups in the molecularstructure, together with an “aglycon” (nitrogen-containing portion),which may be cyclical or acyclical. AGPs having acyclical aglycon groupsare disclosed, for instance, in U.S. Pat. Nos. 6,113,918; 6,303,347 and6,355,257. AGPs having cyclical aglycon groups are disclosed, forinstance, in WO 02/012258.

The above-mentioned documents describe the production of the AGPcompounds by two alternative processes. In one process a protected3-O-acyloxyacylated glycosyl halide containing a phosphonate side chainis coupled with an aminoalkanol or aminoalkanethiol of the typedescribed in the patents. The reaction product is then selectivelyacylated to provide additional acyl groups, as described, and protectinggroups are removed. In the second process, both the phosphonate sidechain and the fatty acid groups are incorporated after the couplingreaction. Additional process information for producing AGP compounds iscontained in Johnson et al., Bioorg. Med. Chem. Lett. 9: 2273 (1999).

Disaccharides that may be produced by the processes described hereininclude components of the well known immunostimulant monophosphoryllipid A (contained, for example in MPL® immunostimulant (Corixa Corp.)Other disaccharides that may be produced are disclosed in, for instance,PCT application WO 01/90129 and U.S. Pat. Nos. 6,013,640; 4,987,237;4,912,094; 4,436,727; and 4,436,728. In U.S. Pat. No. 6,103,640 thedisaccharide was prepared by coupling an N-acyloxyacylated orN-protected glycosyl acceptor unit with a protected and/or3-O-acyloxyacylated glycosyl donor unit. The protecting groups werevariously benzyl (Bn) and 2,2,2-trichloroethoxycarbonyl (Troc) groups.The glycosyl acceptor and donor units were constructed separately usinga series of substituent protection and deprotection steps, beginningwith the known starting materials benzyl- and2-(trimethylsilyl)ethyl-2-amino-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside,respectively.

Glycosyl halides are used in many processes to introduce a glycosidemoiety into a molecule, typically as part of a multistep synthesis inthe field of saccharide chemistry. They are useful intermediates forincorporating a wide variety of groups, typically by reaction withnucleophiles, especially oxygen, sulfur, and nitrogen nucleophiles. Itwould be advantageous to provide a process for producing the AGP anddisaccharide compounds using a glycosyl halide as a starting material.

Various ways of producing glycosyl halides have been described.Generally, they involve halogenation of an existing glycoside (which maycontain typical protecting groups on reactive moieties such as amino orhydroxyl).

In U.S. Pat. No. 6,299,897, for example, an ethyl ester of the glycosidein question (in this instance, N-acetyl neuraminic acid) is reacted withacetyl chloride to produce the corresponding glycosyl chloride. In U.S.Pat. No. 5,843,463, a glycosyl chloride is produced by reacting theglycoside in question(3-O-allyl-5-O-benzyl-1,2-O-methoxybenzylidene-alpha-D-ribofuranose)with trimethylsilyl chloride. The reaction is conducted by mixing thetwo reactants or by dissolving the glycoside in the trimethylsilylchloride.

U.S. Pat. No. 4,613,590 discloses a process for preparation of glycosylchloride by treatment of the glycoside with titanium tetrachloride. InSugiyama et al., Org. Lett. 2: 2713 (2000), glycosyl chlorides wereprepared by reaction of thioglycosides with chlorosulfoniun chloride.

Kovac, Carbohydr. Res. 245: 219(2993) prepared a glycosyl chloride byreaction of the glycoside with dichloromethyl methyl ether and zincchloride. Takeo et al., Carbohydr. Res. 245: 81 (1993) produced aglycosyl chloride by reaction with chlorine. Magnusson et al., J. Org.Chem. 55:3181 (1990) produced a glycosyl chloride by reaction of the2-(timethylsilyl)ethyl glycoside with 1,1-dichloromethyl methyl ether inthe presence of a catalytic amount of zinc chloride.

SUMMARY OF THE INVENTION

This invention relates to a group of related novel processes for theproduction of aminoalkyl glucosaminide phosphates and of disaccharides,together with intermediate processes and compounds.

In one aspect, the invention comprises processes for the production ofaminoalkyl glucosaminide (AGP) compounds.

In a second aspect the invention relates to a process for producingglycosyl halides that comprises reacting a silyl glycoside with adihalomethyl alkyl ether in the presence of zinc chloride, zinc bromide,boron trifluoride, or a similar Lewis acid. This step also comprises thefirst of a two-step process for removing an anomeric silyl protectinggroup from the silyl glycoside by first reacting it to produce aglycosyl halide, which is then reacted it with a silver salt in thepresence of water to produce a hemiacetal.

In another aspect this invention comprises a process comprising firstproducing the glycosyl halide as above, followed by reaction of theglycosyl halide with a monosaccharide in the presence of a silver saltto form a disaccharide.

Another aspect of this invention comprises a process for producing adisaccharide comprising reacting a monosaccharide with a silylglycoside.

Yet another aspect of this invention comprises a process for silylationof a disaccharide and optionally for subsequently adding a phosphonoside chain to the disaccharide.

A still further aspect of this invention comprises a process forproducing a triacylated disaccharide from a disaccharide.

Yet another aspect of the invention is a process for removing an acetylprotecting group from a disaccharide.

Yet a further aspect of this invention comprises a process forproduction of a phosphorylated disaccharide by (a) selectivelyprotecting the 6′-hydroxyl substituent of a disaccharide; and b) addinga phosphono side chain to the disaccharide at the 5′-position.

A still further aspect of this invention comprises a process forsimultaneously removing all silyl-based protecting groups from adisaccharide having a plurality of silyl-based protecting groups.

Other aspects of this invention include other novel processes and novelintermediates, and/or will be apparent from the description thatfollows.

DETAILED DESCRIPTION OF THE INVENTION

Definitions: as used herein:

“Glycoside” refers to a tetrahydropyran ring bearing a substituent atthe 1-position (i.e., at one of the carbon atoms adjacent to the oxygenatom in the ring) that is a hydroxy, optionally substituted alkoxy, ortrisubstituted silyloxy group. Glycosides may also contain substituentsat other positions, typically protected or unprotected hydroxy or aminogroups.

“Silyl glycoside” refers to a glycoside wherein the group attached atthe 1-position is a trisubstituted silyloxy group such as atrimethylsilyloxy, tert-butyldimethylsilyloxy, ortert-butyldiphenylsilyloxy group. The silyl component of this group hasthe formula R_(a)R_(b)R_(c)Si, wherein R_(a), R_(b), and R_(c) areindependently selected from the group consisting of C₁–C₆ alkyl, C₃–C₆cycloalkyl, and optionally substituted phenyl. Preferably one of theR_(a), R_(b), and R_(a) groups is larger than methyl; relativelyhindered groups such as t-butyl, phenyl, and isopropyl are preferred.Included among the silyl components are aryldialkylsilyl,diarylalkylsilyl, and triarylsilyl groups. Typical examples includetriisopropylsilyl, triphenylsilyl, t-butyldimethysilyl (TBS), andt-butyldiphenylsilyl (TBDPS) groups. The silyl component of the silylglycoside is most preferably a TBS or TBDPS group.

The silyl glycoside can generally be represented by the formula (II)

wherein R₂₀ is a trisubstituted silyl group, preferably TBS or TBDPS,and W, X, Y, and Z independently represent H, optionally protectedhydroxy, optionally protected amino, or optionally substituted alkylgroups. Typically, Z represents an optionally protected hydroxymethylgroup.

“Dihalomethyl alkyl ether” refers to a compound bearing an alkoxy groupand two halogen atoms on a single carbon atom. Typical examples includedichloromethyl methyl ether (CHCl₂OCH₃), dichloromethyl ethyl ether(CHCl₂OC₂H₅), dibromomethyl methyl ether (CHBr₂OCH₃), 1,1-dichloroethylethyl ether (CH₃CCl₂OC₂H₅), and the like. Dichloromethyl methyl ether ispreferred in the processes of this invention.

“Glycosyl halide” refers to a 2-halotetrahydropyran compound, forexample, 2-chlorotetrahydropyran or 2-bromotetrahydropyran. Thepreferred halogens are fluoride, chloride, and bromide, with chloridebeing most preferred. In addition, the glycosyl halides used in theprocesses of this invention will have other substituents analogous tothose in formula (II) above.

Glycosyl halides are generally represented by formula (III):

wherein W, X, Y and Z are as defined above for formula (II) and A is Cl,Br, or F.

“Aliphatic” means a straight or branched chain, or non-aromaticcyclical, hydrocarbon radical, or combination thereof, which may befully saturated, or mono- or polyunsaturated and can include di- andmultivalent radicals, having the number of carbon atoms designated (i.e.C₁–C₁₀ means one to ten carbon atoms). Examples of saturated acyclicaliphatic groups (also termed “alkyl” groups) include, but are notlimited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, isobutyl, sec-butyl, homologs and isomers of, for example,n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturatedaliphatic group is one having one or more double bonds or triple bonds.Examples of unsaturated acyclic aliphatic groups include, but are notlimited to, vinyl, 2-propenyl, isopropenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. Examples ofcyclical aliphatic groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, and the like.

Divalent aliphatic groups include saturated and unsaturated groupssimilar to those mentioned above, for example methylene, —CH₂—;ethylene, —CH₂CH₂—; n-butylene, —CH₂CH₂CH₂CH₂—; and unsaturated groupssuch as —CH═CH—, —CH═CH—CH₂CH₂— and the like.

The terms “oxyaliphatic”, “aminoaliphatic” and “thioaliphatic” are usedin their conventional sense, and refer to aliphatic groups attached tothe remainder of the molecule via an oxygen atom, an amino group, or asulfur atom, respectively. “Alkoxy”, “thioalkoxy” and “aminoalkyl” referto such groups containing saturated acyclic aliphatic moieties.

The term “heteroaliphatic,” by itself or in combination with anotherterm, means, unless otherwise stated, a group analogous to an aliphaticgroup, i.e. a saturated or unsaturated straight or branched chain, orcyclic, radical, or combinations thereof, consisting of the statednumber of carbon atoms and further comprising at least one heteroatomselected from the group consisting of O, N, Si and S, and wherein thenitrogen and sulfur atoms may optionally be oxidized and the nitrogenheteroatom may optionally be quaternized. The heteroatom(s) O, N and Sand Si may be placed at any interior position of the heteroaliphaticgroup or at the position at which that group is attached to theremainder of the molecule. Examples include, but are not limited to,—CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, and—CH₂—CH═N—OCH₃.

Aliphatic groups may be substituted or unsubstituted. Substituentsinclude a variety of groups selected from: —OR′, ═O, ═NR′, ═N—OR′,—NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NRR′R″)═NR′″, —NR′C(NR′R″)═NR′″, —NR—C(NR′R″)═NR′″, —S(O)R′,—S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂ in a number ranging fromzero to (2 m′+1), where m′ is the total number of carbon atoms in suchradical. R′, R″ and R′″ each independently may be hydrogen, optionallysubstituted alkyl, aryl optionally substituted with 1–3 halogens,optionally substituted alkoxy, optionally substituted thioalkoxy oroptionally substituted aryl-(C₁–C₄)alkyl groups. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″ and R′″ groups whenmore than one of these groups is present. When R′ and R″ are attached tothe same nitrogen atom, they can be combined with the nitrogen atom toform a 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant toinclude 1-pyrrolidinyl and 4-morpholinyl.

“Aromatic” or “aryl” refers to the typical substituted or unsubstitutednon-aliphatic hydrocarbyl groups of this class, i.e., a polyunsaturated,typically aromatic, hydrocarbon substituent, which can be a single ringor multiple rings (up to three rings) which are fused together or linkedcovalently, such as phenyl, naphthyl, and the like.

“Arylalkyl” refers to alkyl groups subsisted by one or more aryl groups;for instance, benzyl, phenethyl, triphenylmethyl, and the like.

“Acyl” refers to a group derived from an organic acid by removal of thehydroxy group. Acyl compounds may in general be aliphatic, aromatic orheterocyclic in nature. “Aliphatic acyl” refers to such groups derivedfrom saturated or unsaturated aliphatic acids, and includes groups suchas acetyl, propionyl, butyryl, hexanoyl, decanoyl, dodecanoyl,tetradecanoyl, and the like. In defining acyl groups by their carbonatom content, the reference is to the carbon atom content of the entiregroup. Thus, acetyl is a C₂ acyl group; propionyl is a C₃ acyl group,tetradecanoyl is a C₁₄ acyl group, etc.

“Alkanoyloxycarbonyl” refers to groups having a saturated or unsaturatedaliphatic group, or an arylalkyl group such as benzyl, linked through anoxygen atom to a carbonyl group, i.e. a group having the general formulaAlk.-OC(O)— in which Alk. stands for an aliphatic or arylalkyl group asdefined above.

“Alkanoyloxyacyl” refers to a saturated or unsaturated acyl groupsubstituted at the indicated position by an aliphatic group Al.C(O)O— inwhich Al. stands for an acyclic saturated or unsaturated aliphaticgroup. The overall alkanoyloxy group preferably has from 2 to 24 carbonatoms, most preferably from 6 to 14 carbon atoms. The acyl portion ofthe alkanoyloxyacyl group contains from 6 to 14 carbon atoms. A typicalgroup of this type is the 3-(n-alkanoyloxy)acyl group, where the acylgroup is tetradecanoyl and the alkanoyloxy group contains from 2 to 20,preferably from 6 to 14, carbon atoms, inclusive. Similarly “alkanoyl”refers to a group Al.C(O)— wherein Al. is as defined above.

“Protecting group” refers to any of a large number of groups used toreplace one or both hydrogens of a reactive group such as a hydroxy,amino or thiol group, so as to block, prevent, or reduce reactivity ofthe group. Examples of protecting groups (and a listing of commonly usedabbreviations for them) can be found in T. W. Greene and P. G. Futs,“Protective Groups in Organic Chemistry” (Wiley), Beaucage and Iyer,Tetrahedron 48:2223 (1992) and Harrison et al., Compendium of SyntheticOrganic Methods, vols. 1-8 (Wiley).

Representative amino protecting groups include those that form acarbamate or amide with the nitrogen atom, as well as those groupscollectively referred to in the Greene and Futs text as “special —NHprotective groups”. Representative examples of amino protecting groupsinclude acetyl (Ac), trifluoroacetyl, benzyloxycarbonyl (Cbz),tert.-butoxycarbonyl (Boc), allyloxycarbonyl (Aoc),9-fluorenylmethyloxy-carbonyl (Fmoc), nitro-versatryloxycarbonyl (Nvoc),optionally substituted phthaloyl and the like.

Representative hydroxy protecting groups include those where the hydroxygroup is either acylated or alkylated, such as by the formation ofethers or esters using, for instance, acetyl, benzyl, trityl, alkyl,tetrahydropyranyl, allyl and trisubstituted silyl groups.

The choice of a protecting group for a given compound, purpose or set ofconditions is within the skill of those in the art, and is done so as toprotect, generally or selectively, the reactive group in question underthe prevailing conditions (presence of other reactive compounds, pH,temperature, etc.) Protecting groups that may be used in this inventionand are mentioned herein include phthaloyl acetyl (Ac), benzyl (Bn),2,2,2-trichloroethoxycarbonyl (Troc), t-butyldimethylsilyl (TBS),t-butyldiphenylsilyl (TBDPS), and 2,2,2-trichloro-1,1-dimethylethylchloroformyl (TCBOC) groups. As is known in the art, a certainprotecting group or type of group may be more suitable than others foruse with a particular compound or in a given situation, and advantage istaken of these suitabilities in developing processes that involvecompounds with reactive groups such as hydroxy and/or amino. Thus, aswill be seen below, a reaction scheme can be developed for producing orreacting certain compounds in which general or selective protection ordeprotection (removal of protecting groups) is carried out at certainpoints. For instance, in order to selectively react a hydroxy group in acompound that also contains an amino group, or vice versa, the groupwhose reaction is not desired at this point can be blocked with aprotecting group that is not removed under conditions of the reaction(for example, is not base-hydrolyzable if the reaction is to beconducted under basic conditions, while the group to be reacted can beprotected by a group that is base-hydrolyzable, so that said groupbecomes unblocked, and thus reactive, at that time. Similarly as will beseen below, in order to selectively react a group, e.g., a hydroxylgroup, located at one position in the molecule, it may be protected witha different protecting group than other hydroxyls in the molecule. Asused herein, the designation “PG” refers to protecting groups that formesters, ethers or carbonates with hydroxy groups [i.e., with the oxygenatom of a hydroxy group] or that form amides or carbamates with aminogroups [i.e. with the nitrogen atom of an amino group. The designation“PG′” is used herein to refer to optionally substituted phthaloylgroups, for example phthaloyl or tetrachlorophthaloyl, and which may beused to protect an amino group, as shown. However, in any event, theselection of particular protecting groups used or illustrated in theprocesses described herein is not in any way intended to limit theinvention.

The Major Products

The major products produced using the processes and intermediates ofthis invention comprise a group of compounds that include both AGPcompounds, which are mono-saccharides, and disaccharides of somewhatanalogous structure. In general, the products can be depicted by theformulas (I) and (I a–c):

and pharmaceutically acceptable salts and derivatives thereof, wherein Yis —O— or —NH—; R¹ and R² are each independently selected from saturatedand unsaturated (C₂–C₂₄) aliphatic acyl groups; R⁸ is —H or —PO₃R¹¹R¹²,wherein R¹¹ and R¹² are each independently —H or (C₁–C₄)aliphaticgroups; R⁹ is —H, —CH3 or —PO₃R¹³R¹⁴, wherein R¹³ and R¹⁴ are eachindependently selected from —H and (C₁–C₄) aliphatic groups; and whereinat least one of R⁸ and R⁹ is a phosphorus-containing group, but R⁸ andR⁹ are not both phosphorus-containing groups; and X is a group selectedfrom the formulae:

wherein the subscripts n, m, p, q, n′, m′, p′ and q′ are eachindependently an integer of from 0 to 6, provided that the sum of p′ andm′ is an integer from 0 to 6; the subscript r is independently aninteger of from 0 to 14 and may be the same or different; R³, R¹¹, andR¹² are independently saturated or unsaturated aliphatic (C₂–C₂₄) acylgroups; and when X is formula (Ia) or (Ic) one of R¹, R², R³, R¹¹ andR¹² is optionally hydrogen; R⁴ and R⁵ are independently selected from Hand methyl; R⁶ and R⁷ are independently selected from H, OH, (C₁–C₄)oxyaliphatic groups, —PO₃H₂, —OPO₃H₂, —SO₃H, —OSO₃H, —NR₅R₆, —SR¹⁵, —CN,—NO₂, —CHO, —CO₂R¹⁵, —CONR¹⁵R¹⁶, —PO₃R¹⁵R¹⁶, OPO₃R¹⁵R¹⁶, —SO₂R¹⁵ and—OSO₃R¹⁵, wherein R¹⁵ and R¹⁶ are each independently selected from H and(C₁–C₄) aliphatic groups; R¹⁰ is selected from H, CH₃, —PO₃H₂,ω-phosphonooxy(C₂–C₂₄)allyl, and ω-carboxy(C₁–C₂₄)alkyl; R¹³ isindependently selected from H, OH, (C₁–C₄) oxyaliphatic groups,—PO₃R¹⁷R¹⁸, —OPO₃R¹⁷R¹⁸, —SO₃R¹⁷, —OSO₃R¹⁷, —NR¹⁷R¹⁸, —SR¹⁷, —CN, —NO₂,—CHO, —CO₂R¹⁷, and —CONR¹⁷R¹⁸, wherein R¹⁷ and R¹⁸ are eachindependently selected from H and (C₁–C₄) aliphatic groups; and Z is —O—or —S—.The Processes and Intermediates

One process of this invention comprises the production of glycosylhalides that comprises reacting an O-silyl glycoside with a dihalomethylalkyl ether in the presence of zinc chloride, zinc bromide, borontrifluoride, or a similar Lewis acid. More specifically, in thisprocess, a glycosyl halide is formed by reacting a silyl glycosidehaving the formula (II):

wherein R₂₀ is a trisubstituted silyl group having the formulaR_(a)R_(b)R_(c)Si in which R_(a), R_(b) and R_(c) are independentlyselected from the group consisting of C₁–C₆ alkyl, C₃–C₆ cycloalkyl andoptionally substituted phenyl, preferably TBS or TBDPS, and W, X, Y, andZ independently represent H, optionally protected hydroxy, optionallyprotected amino, and optionally substituted alkyl groups, with adihalomethyl alkyl ether, preferably dichloromethyl methyl ether, in thepresence of zinc chloride, zinc bromide, boron trifluoride, or asimilarly suitable Lewis acid. The Lewis acid is used in about astoichiometric amount with respect to the silyl glycoside. The reactionto produce the glycosyl halide is conducted at a temperature of fromabout −30° C. to about 50° C., preferably from about 0° C. to about 30°C., and in the presence of a solvent such as chloroform,dichloromethane, dichloroethane, or similar solvents that are inert tothe conditions required for the reaction. The temperature of thereaction is selected to allow the reactants to substantially dissolveand to prevent the dihalomethyl alkyl ether from boiling away. Yields ofthe desired product glycosyl halide are generally from about 50 to about95%. Selection of such solvents is within the knowledge of those ofordinary skill in the art. The silyl glycosides are producedconventionally, typically in a protected form, as is known in the art.However, certain silyl glycosides, such as certain triacetylated silylglycosides and derivatives thereof, may be produced via novelintermediates described below, which form an aspect of the invention.

One of skill in the art will appreciate that the glycosyl halides mayexist as isomers if other substituents are present on the glycosylhalide ring. The invention includes production of the separate isomersas well as mixtures of both isomers. Conditions for reactions of manynucleophiles with glycosyl halides are well known to persons of ordinaryskill in the art.

The resulting glycosyl halides thus typically have the formula (III)

-   -   wherein A is Cl, Br, or F and W, X, Y and Z are as defined        above.

In one preferred embodiment, the silyl glycoside and resulting productsare substituted at the 3-position (substituent X) by an aliphatic acylgroup, preferably an alkanoyloxyacyl group, more preferably a3-n-alkanoyloxyacyl group, and most preferably a3-alkanoyloxytetradecanoyl group, in which the aliphatic or alkanoylgroup contains from 2 to 24, preferably from 2 to 18, and mostpreferably from 6 to 14, carbon atoms, and the protecting groups in thecompound in question are preferably Troc groups or similaralkanoyloxycarbonyl groups. In such embodiments the compounds have thegeneral formula (IV) or (V):

-   -   wherein A is Cl, Br, or F; R₂₀ is a trisubstituted silyl group        and R₂₁ is an aliphatic acyl group, preferably a        3-n-alkanoyloxytetradecanoyl group. Note that in this formula        and those that follow, protecting groups have been specifically        identified for purposes of illustration and/or clarity. However,        as known in the art, other protecting groups as defined        generally above for “PG” may be used, as suitable. Thus, for        instance, more generally these compounds can be represented by        the formula

where PG represents protecting groups that form an ether, ester orcarbonate with the oxygen atom or that form an amide or carbamate withthe nitrogen atom, respectively.

In another preferred embodiment, the silyl glycoside has a hydroxylgroup at the 4-position substituted with a phosphate ester group such asa dialkylphosphonyl or diarylphosphonyl group, R₂₁ is an alkanoyloxyacylgroup, preferably a 3-n-alkanoyloxytetradecanoyl group, and theprotecting groups are preferably “TCBOC” groups, obtained from2,2,2-trichloro-1,1-dimethylethyl chloroformate, or similaralkanoyloxycarbonyl protecting groups such as Troc; i.e., the silylglycoside may have the specific formula (VI):

-   -   wherein R₂₀ is a trisubstituted silyl group, preferably TBS or        TBDPS; R₂₁ is an aliphatic acyl, preferably an alkanoyloxyacyl,        group; and R₂₂ is alkyl, aryl, or arylalkyl, or may have a more        general formula which allows for the use of other suitable        protecting groups,        and the glycosyl halide correspondingly has the more specific        formula (VII):

-   -   wherein R₂₁ and R₂₂ are as defined above and A is Cl, Br, or F.

In one aspect of the invention the glycosyl chlorides thus produced arereacted with a monosaccharide, preferably in the presence of a silversalt, to produce a disaccharide by this two-step process.Monosaccharides that may be used as reactants in this process include,for example, those having the formulas (i)-(iii):

-   -   wherein R₂₃ is an aliphatic acyl group, preferably a        3-n-alkanoyloxytetradecanoyl group, as described above.

Disaccharides that can be produced by such processes include thosehaving the formulas (iA)–(ivA):

-   -   wherein R₂₁, R₂₂ and R₂₃ are as defined above, and the indicated        protecting groups are exemplary of those that may be used.

Reactions to produce products (iA)–(ivA) according to this invention aregenerally conducted at a temperature of from about −30° C. to about 30°C., in a chlorinated or other solvent in the presence of a silvercatalyst such as silver trifluoromethanesulfonate (triflate) and underanhydrous conditions, with or without other additives such as molecularsieves or buffering agents such as tetramethylurea.

In another aspect of the invention the silyl glycosides of Formula (II)are coupled with a monosaccharide directly, without proceeding throughformation of a glycosyl halide. The resulting product is again adisaccharide having substituents according to the starting materials.Such a process is generally conducted at a temperature of from about−78° C. to about 50° C. in the presence of a suitable Lewis acidcatalyst such as trimethylsilyl triflate of boron trifluoride etheratewith or without the addition of drying or buffering agents. In anotheraspect of this invention protecting groups can in effect be removed froma silyl glycoside having such groups by reacting it with a dihaloalkylether to produce the glycosyl halide, and then reacting the glycosylhalide with a silver salt such as silver oxide or silver carbonate inthe presence of water to produce the corresponding hemiacetal.

The disaccharides produced by either process may be further reacted bysilylating the hydroxyl group at the 4-position of the reducing sugarwith a silylating group such as TBS in the presence of imidazole andN,N-dimethylformamide to produce a 3,4-bis-silylated compound. Additionof a phosphate group in the 4-position of the non-reducing sugar is thenachieved by a sequence of steps involving (1) deprotection of the 4,6protecting groups (typically acetate or Troc), (2)N-deprotection/acylation, (3) selective protection of the primary6-position with a group such as TCBOC, and (4) by reacting the6-protected disaccharide with a phosphonylating agent such as aphosphoramidite reagent, e.g., dibenzyl diisopropylphosphoramidite[providing a dibenzylphosphono side chain], or a chlorophosphate such asbis(2,2,2-trichloroethyl)chlorophosphate [providing abis(2,2,2-trichloroethyl)phosphono side chain] or diphenylchlorophosphate [providing a diphenylphosphono side chain].

The invention also, analogously, includes processes for the productionof triacylated disaccharides such as those having the formula (VIII):

-   -   wherein R₂₁, R₂₃ and R₂₄ are aliphatic acyl, preferably        alkanoyloxyacyl, groups, and R₂₂ is an optionally substituted        alkyl, aryl, or arylalkyl group, by selectively protecting the        C-6 hydroxyl group of a corresponding disaccharide with        2,2,2-trichloro-1,1-dimethylethyl chloroformate in the presence        of a tertiary amine such as pyridine. Preferably, R₂₁, R₂₃ and        R₂₄ are (R)-3-hexadecanoyloxytetradecanoyl,        (R)-3-octadecanoyloxytetradecanoyl, and        (R)-3-tetradecanoyloxytetradecanoyl, respectively, but they can        be the same or different depending on the desired substitutions        and nature of the monosaccharide donor used in the glycosylation        step.

This invention also relates to processes for producing aminoalkyl andcyclic aminoalkyl glucosaminide (AGP) compounds, that is compounds offormula (I) in which X is (Ia) or (Ic), in which both the fatty acid andthe phosphate groups are introduced onto the AGP backbone after theinitial glycosylation (coupling) step. These processes involve the useof novel glycoside triol intermediates which can be selectivelyprotected in the sugar 6-position prior to the introduction of theester- and amide-linked acyloxyacyl residues.

One preferred method of the invention for preparing AGP compounds isshown in Scheme 1 below. Scheme 1 depicts the production of specificcompounds of Formula (Ia) but is intended to serve only as an example ofthis aspect of the invention, as the same or a similar process could beused to produce other compounds of the type of Formula (Ia) as well ascompounds of Formula (Ic).

In this process introduction of the aliphatic acyl, e.g.(R)-3-n-alkanoyloxy-tetradecanoyl, and phosphate groups into theglucosamine and aglycon units is also performed subsequent to thecoupling reaction but, in contrast to the method shown in the prior artpatents, the 3-hydroxyl group is selectively esterified with an alkanoicacid substituted by an aliphatic acyl group, preferably an(R)-3-n-alkanoyloxyalkanoic acid, in the presence of anunprotected/unphosphorylated 4-hydroxyl group with the 6-positionblocked. This is achieved by protecting the 6-hydroxyl group of thesugar unit with a persistent protecting group in lieu of temporaryprotection of the 4,6-hydroxyl positions with an acetonide. Preferably,β-glycoside 8 or the corresponding bis-Troc derivative 9, isde-O-acetylated with a suitable base to give a triol intermediate 10,which is selectively protected on the 6-position with a hindered silylgroup such as t-butyldimethylsilyl (TBS) under standard conditions knownin the art to give silyl-protected intermediate 12. The triolintermediate 10 is a novel compound. 3-O-Acylation of 12 with(R)-3-n-alkanoyloxytetradecanoic acid, for instance, followed bydeprotection/acylation of the sugar and aglycon amino groups,simultaneously (PG=Troc) or sequentially (PO=Aoc), using either zinc(PG=Troc) or zinc and Pd(0) (PG=Aoc) in the deprotection step and(R)-3-n-alkanoyloxytetradecanoic acid in the acylation step, provideshexaacylated intermediate 13. Pentaacylated compounds, i.e. in which oneof the acyl groups R¹, R², R³, R¹¹ or R¹² is hydrogen, can be preparedby utilizing different protecting groups for the two amino groups sothat one or the other can be selectively acylated; for instance, usingan Aoc group for one and a Troc group for the other.

Phosphorylation of the 4-hydroxyl group is carried out by methods knownin the art using preferably either a dibenzyl or di-t-butyl protectedchlorophosphate or phosphoramidite reagent to give the phosphotriester14. The phosphate, silyl and any remaining protecting groups in 14 arethen cleaved under mildly acidic conditions or by other appropriatemeans to give compounds of Formula (Ia). It is important to note thatthe order in which the phosphate and N-linked(R)-3-n-alkanoyloxytetradecanoyl groups in 14 are introduced can bereversed by the appropriate selection of orthogonal phosphate and amineprotecting groups.

A variant of the method shown in Scheme 1 is shown in Scheme 2 andinvolves the use of a commercially available glycosyl donor such as 15possessing either acetyl or phthalimide nitrogen-protecting groups andeither an anomeric acetoxy or halide group. Again, this Scheme isrepresentative of processes of the invention for producing compounds ofFormula (Ia) or (Ic).

In Scheme 2, glycosyl donor 15 is coupled with a similarly N-protectedacceptor unit 16 in the presence of a suitable catalyst to giveβ-glycoside 17. Since N-acetyl and phthalimide groups typically requirestrongly basic conditions for deprotection, the use of a base-stableether-linked protecting group such as triphenylmethyl (trityl, Tr) onthe 6-position is generally required. Accordingly, de-O-acetylation of17 under standard conditions followed preferably by selectivetritylation of the 6-position gives diol 18. Base-induced cleavage ofthe N-acetyl or phthalimide groups followed by simultaneous orsequential N- and O-acylation of the resulting diamino diol intermediatewith an (R)-3-n-alkanoyloxytetradecanoic acid in the presence ofsuitable coupling reagent(s) affords the hexaacylated derivative 19. Thediamino diol intermediate formed by treating compound 18 with base hasthe formula:

Phosphorylation of 19 with a chlorophosphate or phosphoramidite reagentas in Scheme 1 followed by deprotection under mildly acidic conditionsor by other appropriate means gives compounds of Formula (Ia).

In the above Schemes 1 and 2 the various groups R₁–R₇, n, p and q are asdefined above.

The invention is further illustrated by the examples that follow. Theseexamples are presented solely as illustrative of the invention and donot in any way limit its definition or scope.

EXAMPLE 1 Production of2-Deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1,-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranosylChloride

This process is depicted in scheme A, below, and includes novelintermediates (iv), (vi) and (vii), which comprise aspects of thisinvention.

(a) Production of t-Butyldiphenylsilyl2-Deoxy4,6-O-isopropylidene-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranoside(Scheme A, Compound vi).

(1) 2,2,2-Trichloroethoxycarbonyl chloride (200 g, 0.944 mol) is addedportionwise to a solution of D-glucosamine hydrochloride (v, 200 g,0.927 mol) and NaHCO₃ (200 g, 2.4 mol) in water (4 L) in a 10 L 3-neckround-bottom flask and the resulting mixture is mechanically stirredovernight at room temperature. The white precipitate that forms iscollected by filtration using a 2 L fritted funnel washed with ether (2L), and dried at high vacuum for 3 hours to give 297 g (90%) of2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-D-glucose as a whitesolid (MW 354.57).

(2) A solution of2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-D-glucose (297 g, 0.838mol) obtained in (1) above in a mixture of pyridine (1 L, 12.4 mol) andacetic anhydride (1 L, 10.6 mol) in a 10 L round-bottom flask ismechanically stirred at room temperature overnight. The reaction mixtureis concentrated under reduced pressure to give an oil which isazeotroped with toluene (2×1 L) and dried at high vacuum overnight togive 438 g (˜100%; 90% from v) of the tetraacetate as a syrup (MW522.71, TLC (EtOAc) Rf 0.75).

(3) The tetraacetate obtained in (2) above (438 g, 0.838 mol) isdissolved in EtOAc (4 L) and transferred to a 10 L 3-neck round-bottomflask, treated with morpholine (200 mL, 2.29 mol), and mechanicallystirred for 8 hours at room temperature. Reaction completion determinedby TLC (50% EtOAc/hexanes). 3 N aq HCl (2 L) is added and the resultingmixture is stirred for 30 minutes. The mixture is transferred to a 6 Lseparatory funnel and the layers are separated. The organic phase iswashed with saturated aq NaCl (1 L), dried (Na₂SO₄), and concentrated togive 373 g (93%, 84% from v) of the 1-O-deprotected derivative(hemiacetal) as a white foam (MW 480.67; TLC (50% EtOAc/hexanes) Rf0.22).

(4) A solution of hemiacetal obtained in (3) above (373 g, 0.776 mol)and imidazole (132 g, 1.94 mol) in N,N-dimethylformamide (DMF, 430 mL,1.8 M) is treated with t-butylchlorodiphenylsilane (242 mL, 0.931 mol),and stirred for 48 hours at room temperature. Reaction completion isconfirmed by TLC (50% EtOAc/hexanes). The reaction mixture ispartitioned between ethyl ether (4 L) and water (1 L) in a 6 Lseparatory funnel and the layers separated. The ether layer is washedwith water (1 L), dried (Na₂SO₄), and concentrated to give abronze-colored oil which is crystallized from EtOAc-hexanes (˜1:2 v/v)in three crops to provide 474 g (85%, 71% from v) of thet-butyldiphenylsilyl glycoside iv as a white solid (MW 719.08; TLC (50%EtOAc/hexanes) Rf 0.44).

(5) A solution of the silyl glycoside obtained in (4) above (474 g,0.659 mol) in MeOH (2 L) in a 3 L 3-neck round-bottom flask is treatedwith ammonium hydroxide (300 mL, 4.5 mol) (some precipitation occurs)and stirred at room temperature overnight, and then treated with asecond portion of ammonium hydroxide (50 mL, 0.75 mol) and again stirredovernight. Reaction completion is determined by TLC (EtOAc). Thereaction mixture is concentrated and the resulting residue is dissolvedin EtOAc (500 mL), placed on a pad of silica gel (1 kg) in a 3 L frittedglass funnel, and eluted with 50% EtOAc-hexanes (5 L) and EtOAc (7 L).The fractions containing the product are concentrated in a 3 Lround-bottom flask to give 329 g (84%, 60% from v) of the triol (MW592.97, TLC (EtOAc) Rf 0.35).

(6) A slurry of the triol obtained in (5) above (329 g, 0.555 mol) in2,2-dimethoxypropane (1.5 L) in a 3 L round-bottom flask is treated withcamphorsulfonic acid (6.4 g, 0.028 mol) and magnetically stirred at roomtemperature overnight, giving a light yellow solution. Solid NaHCO₃ (4.6g, 0.055 mol) is added and the resulting mixture is stirred for 2 hoursat room temperature and then concentrated to dryness. The crude productobtained is dissolved in dichloromethane (1.2 L), divided into two equalportions, and placed on silica gel (1 kg, pre-wetted with 30%EtOAc/hexanes) in two separate 3 L fritted glass funnels, and elutedwith 30% EtOAc/hexanes (10 L) and 50% EtOAc/hexanes (8 L). Fractionscontaining purified product are combined and concentrated to givecompound vi as an amorphous solid. The product can be further purifiedby crystallization from hexanes, if necessary.

-   -   Molecular Formula: C₂₈H₃₆Cl₃NO₇Si    -   Molecular Weight: 633.04    -   Theoretical Yield: 587 g (based on v)    -   Expected Yield: 306 g (87%, 52% from v)    -   TLC: Rf 0.60 (EtOAc)        (b) Production of t-Butyldiphenylsilyl        2-Deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranoside        (Scheme A, Compound vii).

(1) A solution of compound vi (141 g, 0.223 mol) in CH₂Cl₂ (1 L) in a 2L round bottom flask is treated with3-(R)-(tetradecanoyloxy)tetradecanoic acid (101.7 g, 0.224 mol), DCC (55g, as a melt, 0.267 mol) and 4pyrrolidinopyridine (3.3 g, 0.022 mol),and stirred at room temperature overnight. Reaction completion isdetermined by TLC (20% EtOAc/hexanes). The reaction mixture is filtered,concentrated to ca. one-half volume, divided into two equal portions,and placed on silica gel (1 kg, pre-wetted with 2.5% EtOAc/hexanes) intwo separate 3 L fritted glass funnels. Gradient elution with 2.5%, 5%,and 10% EtOAc/hexanes (8 L each) and concentration of the fractionscontaining the product in a 3 L round-bottom flask gives 220 g (92%) ofthe ester (MW 1069.72, TLC (20% EtOAc/hexanes) Rf 0.53).

(2) The ester obtained in (1) above (218 g, 0.204 mol) is suspended in90% aq AcOH (1 L) in a 3 L round-bottom flask and stirred (on rotaryevaporator) at 70° C. for 2.5 hours, giving a milky solution. Reactioncompletion is determined by TLC (20% EtOAc/hexanes). The reactionmixture is concentrated and residual AcOH is removed azeotropically withtoluene (2×500 mL). The crude product obtained is dissolved in 10%EtOAc/hexanes (400 mL), divided into two equal portions, and placed onsilica gel (1 kg) in two separate 3 L fritted glass funnels. Gradientelution with 10% EtOAc/hexanes (10 L) and 15%, 20%, and 30%EtOAc/hexanes (5 L each) and concentration of the fractions containingthe product gives 193 g (92%, 85% from vi) of the diol (MW 1029.66, TLC(20% EtOAc) Rf 0.10) containing a small amount (<5% by TLC) of6-O-acetyl by-product (Rf 0.25). (Note: The 6-acetate by-product isreadily separated by radial compression chromatography as the4-diphenylphosphate derivative in step (3) below.)

(3) A magnetically stirred solution of the diol obtained in (2) above(193 g, 0.187 mol) in CH₂Cl₂ (1 L) at 0° C. is treated with pyridine(18.2 mL, 0.225 mol) followed by 1,1-dimethyl-2,2,2-trichloroethylchloroformate (49.5 g, 0.206 mol). Progress of the reaction is monitoredby TLC (20% EtOAc/hexanes). Once the reaction is completed by TLC(typically 30-60 minutes, but longer reaction times may be required),triethylamine (55 mL, 0.39 mol), 4-pyrrolidinopyridine (13.9 g, 0.094mol), and diphenyl chlorophosphate (58.2 mL, 0.281 mol), are addedsequentially and the resulting mixture is stirred at room temperatureovernight. Reaction completion is determined by TLC (20% EtOAc/hexanes).The reaction mixture is concentrated to dryness and the residue obtainedis partitioned between EtOAc (1.5 L) and 1.2 N aq HCl (2 L) in a 6 Lseparatory funnel and the layers separated. The EtOAc layer is washedwith water (2 L), dried (Na₂SO₄), and concentrated. The residue obtainedis dissolved in 10% EtOAc/hexanes (500 mL) and purified by gradientelution on a Biotage 150 Hi system (1SOL column) with 10% EtOAc/hexanes(50 L), collecting 950 mL fractions. The fractions containing compoundvii are combined and concentrated.

-   -   Molecular Formula: C₇₀H₉₈Cl₆NO15PSi    -   Molecular Weight: 1465.30    -   Theoretical Yield: 326.8 g (based on vi)    -   Expected Yield: 211 g (77%, 65% from vi)    -   TLC: Rf 0.47 (20% EtOAc/hexanes)        (c) Production of        2-Deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyloxy-tetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranosyl        Chloride (Scheme A, Compound viii).

A solution of compound vii (192 g, 0.131 mol) in CHCl₃ (2 L) at 0° C. ina 5 L round-bottom flask is treated with α,α-dichloromethyl methyl ether(78 mL, 0.87 mol), followed by ZnCl₂ (1.0 M in ether, 100 mL, 0.1 mol)dropwise via an addition funnel. The cold bath is removed and theresulting mixture is stirred at room temperature overnight. Reactioncompletion is determined by TLC (20% EtOAc/hexanes). The reactionmixture is treated with cold saturated aq NaHCO₃ (1 L), stirred for 1hour, and the layers are separated in a 6 L separatory funnel. Theorganic layer is dried (MgSO₄) and concentrated. The residue obtained ispurified on a Biotage 150 Hi system (150 L column) eluting with 10%EtOAc/hexanes (80 L, 950 mL fractions). The fractions containing pureproduct are combined and concentrated.

-   -   Molecular Formula: C₅₄H₇₉Cl₇NO₁₄P    -   Molecular Weight: 1245.36    -   Theoretical Yield: 163.2 g    -   Expected Yield: 141 g (86%)    -   TLC: Rf 0.42 (20% EtOAc/hexanes)

EXAMPLE 2 Preparation of(N-[(R)-3-Decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-O-D-glucopyranosyl]-L-serineTriethylammonium Salt

[a compound of Formula (Ia) in which R₁═R₂═R₃=n-C_(g)H₁₉CO, Z=Y═Y═O,n=m=p=q=0, r=10, R₄═R₅═R₇═R₉═H, R₆═CO₂H, R₈═PO₃H₂)], namely

This example utilizes a process as shown in Scheme 1.

(1) A solution of1,3,4,6-tetra-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxy-carbonylamino)-β-D-glucopyranoside(5.33g, 10.2 mmol) and benzyl N-(2,2,2-trichloroethoxycarbonyl)-L-serine(4.16 g, 11.2 mmol) in anhydrous CH₂Cl₂ (15 mL) was treated dropwisewith boron trifluoride etherate (2.59 mL, 20.4 mmol) and then stirred atroom temperature for 2 h. The reaction mixture was quenched withsaturated aq. NaHCO₃ (20 mL) and the layers separated. The aqueous layerwas extracted with CHCl₃ (2×10 mL) and the combined organic layers werewashed with H₂O (10 mL), dried (Na₂SO₄), and concentrated in vacuo.Flash chromatography on silica gel (gradient elution, 20→50%AcOEt/hexanes) afforded 7.42 g (87%) ofN-(2,2,2-trichloroethoxycarbonyl)-O-[3,4,6-tetra-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino).-β-D-glucopyranosyl]-L-serinebenzyl ester as a white solid (compound 9; X═O, n=m=n=p=q=0, r=10,R₄═R₅═R₇═H, R₆═CO₂Bn).

(2) A solution of the compound prepared in (1) above (408 mg, 0.49 mmol)in tetrahydrofuran (TF; 20 mL) was hydrogenated in the presence of 10%palladium on carbon (30 mg) at room temperature and atmospheric pressurefor 3 h. The reaction mixture was filtered through Celite and thefiltrate was concentrated in vacuo. Flash chromatography on silica gelwith 2% MeOH-CHCl₃ followed by 10% MeOH-CHCl₃ afforded 347 mg (98%) ofN-(2,2,2-trichloroethoxycarbonyl)-O-[3,4,6tetra-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosyl]-L-serineas a white solid. (compound 9; X═O, n=m=p=q=0, r=10, R₄═R₅═R₇═H,R₆═CO₂H)

(3) A solution of the compound prepared in (2) above (998 mg, 1.34 mmol)in methanol (15.5 mL) was treated with ammonium hydroxide (0.21 mL, 5.37mmol) at room temperature for 16 h, followed by additional ammoniumhydroxide (0.21 mL, 5.37 mmol) for 24 h. The reaction mixture wasconcentrated in vacuo and to give a white solid. A suspension of thewhite solid in CH₂Cl₂ (33.5 mL) was treated with benzyl bromide (0.80mL, 6.7 mmol), tetrabutylammonium bromide (432 mg, 1.34 mmol) andsaturated NaHCO₃ (33.5 mL) and the resulting biphasic mixture wasstirred vigorously at room temperature for 24 h and the layersseparated. The aqueous layer was extracted with CHCl₃ (2×15 mL) and thecombined organic layers were washed with H₂O (10 mL), dried (Na₂SO₄),and concentrated in vacuo. The resulting residue was dissolved inanhydrous pyridine (10 mL), treated with t-butyldimethylsilyl chloride(242 mg, 1.61 mmol), and stirred at room temperature for 1.5 h. Thereaction mixture was treated with additional t-butyldimethylsilylchloride (242 mg, 1.61 mmol) and stirred 1.5 h. The reaction mixture waspartitioned between CHCl₃ (10 mL) and H₂O (10 mL). The aqueous layer wasextracted with CHCl₃ (2×15 mL) and the combined organic layers werewashed with H₂O (15 mL), dried (Na₂SO₄) and concentrated in vacuo. Flashchromatography on silica gel using gradient elution (1.0→1.25%CH₃OH/CHCl₃) afforded 724 mg (66%) ofN-(2,2,2-trichloroethoxycarbonyl)-O-[6-O-t-butyldimethylsilyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino).-β-D-glucopyranosyl]-L-serinebenzyl ester as a white solid. (compound 12 PG=Troc, X═O, n=m=p=q=0,r=10, R₄═R₅═R₇═H, R₆═CO₂H)

(4) A solution of the compound prepared in (3) above (892 mg, 1.09 mmol)in anhydrous CH₂Cl₂ (10.5 mL) was treated with(R)-3-decanoyloxytetradecanoic acid (476 mg, 1.20 mmol),1-(3dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDC-MeI; 355mg, 1.20 mmol), and 4-pyrrolidinopyridine (8 mg, 0.054 mmol) at 0° C.for 1 h. The reaction mixture was treated with additional(R)-3-decanoyloxytetradecanoic acid (60 mg) and EDC.MeI (60 mg) at 0°C., stirred 30 min, and concentrated in vacuo. Flash chromatography onsilica gel with 1:6 AcOEt-hexanes afforded 1.10 g (85%) ofN-(2,2,2-trichloroethoxy-carbonyl)-O-[6-O-t-butyldimethylsilyl-3-O-[(R)-3-decanoyloxytetradecanoyl]-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino).-β-D-glucopyranosyl]-L-serinebenzyl ester as a colorless oil.

(5) A solution of the compound prepared in (4) above (1.162 g, 0.967mmol) in 20% aq. THF (16 mL) was treated with zinc dust (632 mg, 9.67mmol) and acetic acid (0.12 mL, 2.13 mmol) and stirred for 1 h at roomtemperature. The reaction mixture was filtered through Celite and thefiltrate concentrated in vacuo. The resulting off-white solid wasdissolved in CHCl₃ (15 mL) and washed successively with 15 mL portionsof 0.1M HCl, saturated aq NaHCO₃, and H₂O. The organic layer was dried(Na₂SO₄) and concentrated in vacuo and the resulting residue was driedovernight under high vacuum. A solution of the residue in anhydrousCH₂Cl₂ (9.5 mL) was treated with (R)-3-decanoyloxytetradecanoic acid(848 mg, 2.13 mmol) and EDC.MeI (632 mg, 2.13 mmol) and stirred at roomtemperature for 2 h. The reaction mixture was concentrated in vacuo andthe residue obtained purified by flash chromatography on silica gel(gradient elution; 20→25% AcOEt/hexanes) to give 1.03 g (66%) ofN-[(R)-3-decanoyloxytetradecanoyl]-O-[6-O-t-butyldimethylsilyl-2-deoxy-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinebenzyl ester as a glassy solid. (compound 13 R₁═R₂═R₃═n-C₉H₁₉CO, X═O,n=m=p=q=0, r=10, R₄═R₅═R₇═H, R₆═CO₂Bn).

(6) A solution of the compound prepared in (5) above (112 mg, 0.069mmol) in anhydrous dichloromethane (1 mL) under argon was treated withdibenzyl diisopropyl phosphoramidite (39 μL, 0.12 mmol) and tetrazole(12 mg, 0.173 mmol) and stirred at room temperature for 1 h. Thereaction mixture was cooled to 0° C. and treated with m-chloroperbenzoicacid (m-CPBA; 33 mg, 0193 mmol) for 30 min. The reaction mixture wasquenched by addition of saturated aq NaHCO₃ (5 mL) and stirred at roomtemperature for 15 min. The aqueous layer was extracted with chloroform(3×5 mL) and the combined organic layers were washed with water (5 mL),dried (Na₂SO₄), and concentrated in vacuo. Flash chromatography with 25%AcOEt-hexanes gave partially purified product which wasrechromatographed on silica gel with 20% AcOEt-hexanes to give 122 mg(93%) ofN-[(R)-3-decanoyloxytetradecanoyl]-O-[6-O-t-butyldimethylsilyi-2-deoxy-4-O-diphenylphosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinebenzyl ester as a colorless oil.

(7) A solution of the compound prepared in (6) above (232 mg, 0.124mmol) in anhydrous THF (10 mL) was hydrogenated in the presence of 20%palladium hydroxide on carbon (46 mg) at room temperature andatmospheric pressure for 36 h. The reaction mixture was filtered throughCelite and the filtrate concentrated under vacuum. The resulting oil(181 mg) was dissolved in CH₂Cl₂ (2.5 mL) and treated withtrifluoroacetic acid (29 μL) and stirred under argon at room temperaturefor 18 h. The reaction mixture was concentrated and co-evaporated withhexanes (2×5 mL). Flash chromatography on silica gel withchloroform-methanol-water-triethylamine (gradient elution;87:12:0.5:0.5→77:22.5:0.5:0.5) afforded 102 mg (55%) ofN-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinetriethylammonium salt (RC-527) as a colorless solid.

EXAMPLE 3 Preparation of(S)-2-[(R)-3-Hexanoyloxytetradecanoylamino]-3-phosphonooxypropyl2-Deoxy-4-O-phosphono-3-O-[(R3-hexanoyloxytetradecanoyl]-2-[(R)-3-hexanoyloxytetradecanoylamino]-β-D-glucopyranosideBis(triethyl)ammonium Salt

[a compound of Formula (I) in which X is (Ia), namelyR₁═R₂═R₃=n-C₅H₁₁CO, Z=Y═O, n=m=p=q=0, r=10, R₄═R₅═R₇═R₉═H, R₆═CH₂OPO₃H₂,R₈═PO₃H₂], namely:

This example utilizes a process as shown in Scheme 1.

(1) In the same manner as described in Example 2-(3),1,3,4,6-tetra-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranoside(0.62 g, 1.18 mmol) and(S)-2-(2,2,2-trichloroethoxycarbonylamino)-3-benzyloxy-1-propanol (0.46g, 1.30 mmol) were coupled in the presence of boron trifluoride etherate(0.3 mL, 2.4 mmol) to give(R)-2-(2,2,2-trichloroethoxycarbonylamino)-3-benzyloxy-1-propyl2-deoxy-3,4,6-tetra-O-acetyl-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas a light yellow solid. (compound 9; X═O, n=m=p=q=0, r=10, R₄═R₅═R₇═H,R₆═CH₂OBn). A solution of this compound in methanol (15 mL) was treatedwith ammonium hydroxide (0.21 mL, 5.37 mmol) at room temperature for 19h, followed by additional ammonium hydroxide (0.20 mL, 5.1 mmol) for 25h. The reaction mixture was concentrated in vacuo and to give a whitesolid. Flash chromatography on silica gel (gradient elution 5→6%CH₃OH/CHCl₃) afforded 0.57 g (63%) of3-benzyloxy-(R)-2-(2,2,2-trichloroethoxycarbonylamino)propyl2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranoside as aglassy solid.

(2) A solution of the compound prepared in (2) above (0.57 g, 0.83 mmol)in anhydrous pyridine (8.5 mL) was treated with t-butyldimethylsilylchloride (0.15 g, 0.99 mmol) and stirred at room temperature for 1.5 h.Additional t-butyldimethylsilyl chloride (0.15 g, 0.99 mmol) was addedand after another 1.5 h the reaction mixture was partitioned betweenCHCl₃ (10 mL) and H₂O (10 mL) and the layers separated. The aqueouslayer was extracted CHCl₃ (2×10 mL) and the combined organic layers werewashed with H₂O (10 mL), dried (Na₂SO₄), and concentrated in vacuo.Flash chromatography on silica gel (gradient elution; 80:1→60:1CHCl₃/CH₃OH) afforded 0.65 g (98%) of3-benzyloxy-(R)-2-(2,2,2-trichloroethoxycarbonylamino)propyl6-O-t-butyldimethylsilyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas a white solid.

(3) In the same manner as described in Example 2-(4), the compoundprepared in (2) above (0.47 g, 0.59 mmol) was acylated with(R)-3-hexanoyloxytetradecanoic acid (0.22 g, 0.64 mmol) in the presenceof EDC-MeI (0.21 g, 0.70 mmol) and 4-pyrrolidinopyridine (4 mg, 0.03mmol) to afford 0.58 g (88%) of3-benzyloxy-(R)-2-(2,2,2-trichloroethoxy-carbonylamino)propyl6-O-t-butyldimethylsilyl-3-O-[(R)-3-hexanoyloxytetradecanoyl]-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas a colorless oil.

(4) In the same manner as described in Example 2-(5), the compoundprepared in (3) above (0.58 g, 0.51 mmol) was deprotected with zinc(0.34 g, 5.14 mmol) and acylated with (R)-3-hexanoyloxytetradecanoicacid (0.39 g, 1.13 mmol) in the presence of EDC.MeI (0.34 g, 1.13 mmol)to afford 0.41 g (56%) of3-benzyloxy-(R)-2-[(R)-3-hexanoyloxy-tetradecanoylamino]propyl6-O-t-butyldimethylsilyl-3-O-[(R)-3-hexanoyloxytetradecanoyl]-2-deoxy-2-[(R)-3-hexanoyloxytetradecanoylamino]-β-D-glucopyranosideas a colorless oil (compound 13 R₁═R₂═R₃═n-C₅H₁₁CO, X═O, n=m=p=q=0,r=10, R₄═R₅═R₇═H, R₆═CH₂OBn).

(5) A solution of the compound prepared in (4) above (0.41 g, 0.29 mmol)in THF (18 mL) was hydrogenated in the presence of palladium hydroxide(0.04 g) at room temperature and atmospheric pressure for 17 h. Thereaction mixture was filtered through Celite, and the filtrateconcentrated in vacuo. Flash chromatography on silica gel (gradientelution; 1:2→1:8 ethyl acetate/heptane) provided 0.3 g (77%) of3-hydroxy-(R)-2-[(R)-3-hexanoyloxytetradecanoylamino]propyl6-O-t-butyldimethylsilyl-3-O-[(R)-3-hexanoyloxytetradecanoyl]-2-deoxy-2-[(R)-3-hexanoyloxytetradecanoylamino]-β-D-glucopyranosideas a colorless oil (compound 13 R₁═R₂═R₃═n-C₅H₁₁CO, X═O, n=m=p=q=0,r=10, R₄═R₅═R₇═H, R₆═CH₂OH).

(6) In the same manner as described in Example 2-(6), the compoundprepared in (5) above (0.30 g, 0.22 mmol) was phosphorylated withdibenzyl diisopropylphosphoramidite (0.25 mL, 0.75 mmol), tetrazole(0.08 g, 1.11 mmol), and m-CPBA (0.33 g, 1.95 mmol) to give 0.30 g (73%)of3-dibenzylphosphonooxy-(R)-2-[(R)-3-hexanoyloxy-tetradecanoylamino]propyl4-dibenzylphosphono-6-O-t-butyldimethylsilyl-3-O-[(R)-3-hexanoyloxytetradecanoyl]-2-deoxy-2-[(R)-3-hexanoyloxytetradecanoylamino]-β-D-glucopyranosideas a colorless oil.

(7) A solution of the compound prepared in (6) above (302 mg, 0.16 mmol)in anhydrous THF (13 mL) was hydrogenated in the presence of 20%palladium hydroxide on carbon (60 mg) at room temperature andatmospheric pressure for 27 h. The reaction mixture was filtered throughCelite and the filtrate concentrated in vacuo. A solution of theresulting oil (226 mg) in CH₂Cl₂ (3.5 mL) was treated withtrifluoroacetic acid (0.04 mL, 0.49 mmol) and stirred under argon atroom temperature for 16 h. The reaction mixture was concentrated andco-evaporated with hexanes (2×5 mL), and the resulting residue driedunder high vacuum to give crude product (226 mg). A portion of the crudeproduct (102 mg) was dissolved in 1:2 CHCl₃/CH₃OH (9 mL), loaded onto aDEAE-cellulose column (15 g, fast flow, Sigma), and eluted with 2:3:1CHCl₃:CH₃OH:H₂O using a 0 to 0.1 M NH₄OAc salt gradient. The fractionscontaining purified product were combined, washed with 0.1 N aq HCl, andconcentrated in vacuo. The residue obtained was lyophilized from 1% aqtriethylamine (pyrogen free) to give 82 mg (81%)(S)-2-[(R)-3-hexanoyloxytetradecanoylamino]-3-phosphonooxypropyl 2-deoxy4-O-phosphono-3-O-[(R)-3-hexanoyloxytetradecanoyl]-2-[(R)-3-hexanoyloxytetradecanoylamino]-β-D-glucopyranosidebis(triethyl)ammonium salt as a white powder: positive FAB-MS calcd for[M+Na]⁺1407.8534, found 1407.8689; ¹H NMR (CDCl₃/CD₃OD): δ (ppm)5.23–5.16 (m, 4H), 4.67 (d, 1H), 4.38 (dd, 1H), 4.19–3.83 (m, 7H), 3.49(m, 2H), 3.06 (m, 12H), 2.64–2.21 (m, 12H), 1.58–1.56 (m, 12H), 1.23 (m,94H), 0.88–0.87 (m, 18H). ¹³C NMR (CDCl₃/CD₃OD): δ (ppm) 173.7, 173.3,173.2, 170.3, 170.1, 100.0, 74.6, 74.0, 70.9, 70.8, 70.3, 66.6, 63.5,60.4, 54.2, 45.8, 41.1, 40.7, 39.3, 34.4, 34.3, 31.9, 31.3, 29.7, 29.4,25.3, 24.7, 22.7, 22.3, 14.1, 13.9, 8.5.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A compound having the formula:

wherein the subscripts n, m, p, and q are each independently an integerof from 0 to 6; R⁴ and R⁵ are independently selected from H and methyl;PG is allyloxycarbonyl (Aoc) or 2,2,2-trichloroethoxycarbonyl (Troc); R⁶and R⁷ are independently selected from H, OH, (C₁–C₄) oxyaliphaticgroups, —PO₃H₂, —OPO₃H₂, —SO₃H, —OSO₃H, —NR¹⁵R¹⁶, —SR¹⁵, —CN, —NO₂,—CHO, —CO₂R¹⁵, —CONR¹⁵R¹⁶, —PO₃R¹⁵R¹⁶, —OPO₃R¹⁵R¹⁶, —SO₃R¹⁵ and—OSO₃R¹⁵, wherein R¹⁵ and R¹⁶ are each independently selected from H and(C₁–C₄) aliphatic groups and X is O or S.
 2. A compound having theformula:

wherein the subscripts n, m, p. and q are each independently an integerof from 0 to 6; R⁴ and R⁵ are independently selected from H and methyl;PG is independently acetyl (Ac) or optionally substituted phthaloyl; R⁶and R⁷ are independently selected from H, OH, (C₁–C₄) oxyaliphaticgroups, —PO₃H₂, —OPO₃H₂, —SO₃H, —OSO₃H, —NR¹⁵R¹⁶, —SR¹⁵, —CN, —NO₂,—CHO, —CO₂R¹⁵, —CONR¹⁵R¹⁶, —PO₃R¹⁵R¹⁶, —OPO₃R¹⁵R¹⁶, —SO₃R¹⁵ and—OSO₃R¹⁵, wherein R¹⁵ and R¹⁶ are each independently selected from H and(C₁–C₄) aliphatic groups; and X is O or S.